Open Access Journal Volume: 1.

Science World Journal of Pharmaceutical Sciences

Studies on In Vitro and In Vivo Antioxidant Evaluation and Total Phenolic,
Flavonoidal Content Estimation of Beta vulgaris root

*Nwaogwugwu CJ1 1
Department of Biochemistry, Faculty of Biological and Physical Sciences,
Nosiri CI
Abia State University, PMB 2000, Uturu, Nigeria
Atasie OC 2
Department of Medical Laboratory Sciences, Imo State University, Owerri
Nwadike C2

Article Type: Research Article
Journal Type: Open Access
Volume: 1 Issue: 1
Manuscript ID: SWJPS-1-105
Publisher: Science World Publishing
Received Date: 18 April 2019
Accepted Date: 02 May 2019
Published Date: 20 May 2019
Article Information
*Corresponding author: Citation: Nwaogwugwu CJ, (2019),
Studies on In Vitro and In Vivo Antioxidant
Nwaogwugwu CJ
Evaluation and Total Phenolic, Flavonoidal
Department of Biochemistry Content Estimation of Beta vulgaris root. Sci
Faculty of Biological and Physical World J Pharm Sci, 1(1); 1-5
Sciences, Abia State University
PMB 2000, Uturu, Nigeria

Copyright: © 2019, Nwaogwugwu CJ, This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0
International License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source
are credited.

ABSTRACT
Beta vulgaris root extracts are traditionally used as a folk medicine to treat ailments such as hypertension, hypoglycemic, inflammatory,
and hepertocellular disorder. In this study the antioxidant potential of root extract of B. vulgaris were mentored in vitro against 2, 2-diphenyl-1-
picrylhydrazyl (DPPH) and modulatory effect on rat brain enzymes. Total phenolic, flavonoid contents, and antioxidant potential against DPPH
of the root extracts at varying concentrations were evaluated using ethyl acetate. Thus concentration of root extracts of B. vulgaris root at 100,
200, and 400 mg/kg body weights were administered orally for (7) days to albino rats followed by evaluated of oxidative stress markers such
as thiobarbituric acid reactive substances (TBARS), Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione (GSH) in the animals brain
homogenate. The percentage inhibition and IC50 value of the root extracts were significantly (P < 0.01) dose dependent compared to ascorbic
acid standard. Similarly SOD, CAT, and GSH increased significantly (P < 0.01) in test animals administered 200 and 400 mg/kg of ethyl acetate
extract of B. vulgaris root. The results obtained here suggest that B. vulgaris roots extracts suggests a neuroprotective potential of this plant
roots.

KEYWORDS: Beta vulgaris, Flavonoids, 2, 2-diphenyl-1-picrylhydrazyl, Oxidative stress, Antioxidant

INTRODUCTION
Oxidation is very essential to many living organisms for the production of energy to fuel biological processes and lead to production of
both free radicals and Reaction Oxygen Species (ROS). These ROS and free radicals are byproduct of normal cellular metabolism of the body
and are produced during irradiation (Ultraviolet [UV] light, X-rays, and γ-rays), inflammation, pollution, and mitochondria-catalyzed electron
transport reactions [1]. ROS may contribute to oxidative damage resulting to loss of function, cellular death including various numbers of
disorders such as coronary atherosclerosis, ischemia, ageing, diabetes, cancer, immunosuppression, and neurodegenerative disorders [2]. The
interruption of the normal cellular function of lipids, proteins, carbohydrates, enzymes, and nucleic acids is due to an imbalance between pro-
oxidants and antioxidants giving rise to oxidative stress [3]. However, the body can form defense against harmful effect of oxygen and nitrogen
reactive species using exogenous and endogenous antioxidant enzymes such as Catalase (CAT), glutathione peroxidase, and Superoxide
Dismutase (SOD); and Non-enzymatic Systems as Thiol Reduced (Glutathione [GSH]), vitamins, minerals, and polyphenols [4]. Antioxidants are
vital substances that possess the ability to protect the body from damage caused by free radical induced oxidative stress.
Beta vulgaris (beet) is a plant which is included in Betoideae subfamily in the Amaranthaceous family. It is the economically most
important crop of the large order Caryophyllales. It has several cultivar groups such as: the root vegetable known as the beetroot or garden
beet; the leafy vegetables chard, spinach beet; and mangelwurzel, which is a fodder crop [16]. Three subspecies are typically recognized. All
cultivars fall into the subspecies B. vulgaris subsp. vulgaris. The wild ancestor of the cultivated beets is the sea beet (B. vulgaris subsp. maritima).
The claimed therapeutic use of beetroot includes its antitumor, carminative, emmenagogue, and hemostatic and renal protective properties
and is a potential herb used in cardiovascular conditions [8]. The juice of beetroot is also consumed as a natural remedy for sexual weakness
and to expel kidney and bladder stones. In recent years, beetroot has gained popularity to be a natural food to boost the energy in athletes
[8]. Recent reports indicate that B. vulgaris extracts (root) possess antihypertensive, hypoglycemic, antioxidant [9], anti-inflammatory, and

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hepatoprotective activities [6]. Previously, red beetroot extract has of tissue homogenate. Changes in absorbance of the reaction solution
been demonstrated to be an effective multiorgan tumor suppressing at 570 nm were determined after 1 min. Results were expressed in
agent in laboratory animals [7]. units/mg protein.
Recent studies have also postulated that renal inflammation, Reduced Glutathione Assay [25]
which is characterized by infiltration of inflammatory cells such
This was estimated by using dithiobisnitro-benzoate as a
as monocytes/macrophages and subsequent release of pro-
substrate. The yellow color developed and read immediately at an
inflammatory cytokines and activation of NF-𝜅B (nuclear factor
absorbance of 412 nm and expressed as μM GSH/g protein.
kappa-light-chain-enhancer of activated B cells) in response to
oxidative stress, is involved in this process phe. This research is Statistical Analysis
therefore aim at evaluate the in vitro and in vivo antioxidant and total
The values were expressed in mean ± standard error of the
Phenolic, Flavonoidal content estimation of B. vulgaris root
mean. Statistical analysis was done by one-way ANOVA followed by
Dunnett’s multiple comparison test versus control. P < 0.05 and P <
MATERIALS AND METHODS 0.01 were considered as significant.
Plant Materials and Preparation of Extract
Fresh roots of Beta vulgaris (1 kg) was macerated and soaked
RESULTS
in 70% ethanol (1 litre) for three (3) days. The obtained root extract Qualitative Phytochemical Screening
was concentrated using rotary evaporator before complete drying.
Phytochemical analysis of extracts of B. vulgaris root revealed
The extract was then resuspended in distilled water (1L).
the presence of alkaloids, glycosides, flavonoids, coumarins, lignins,
Experimental Animals tannins, terpenoids, carbohydrates, protein, fatty acids, and phenolic
compounds.
Thirty six (36) weaned male albino rats of the same stock were
obtained from the animal farm of Imo State University Owerri. The Estimation of Total Phenolics and Flavonoids Contents
animals were taken to the laboratory where they were housed in a
TPC of CEBVR, MEBVR, and EABVR was observed at 8.43 ± 1.03
plastic cage, placed on commercial rat and allowed free access to
mg, 11.59 ± 1.31 mg, and 15.64 ± 1.31 mg of GAE/100 g, respectively,
water ad libitum. The animals were of the same sex, age and weighed
whereas the TFC of CEBVR, MEBVR, and EABVR was observed at
about 160 on average. The animals were subsequently allowed to
0.75 ± 0.10 mg, 1.31 ± 0.01 mg, and 1.97 ± 0.47 mg of QUE/100 g,
acclimatize for two (2) weeks before commencement of experiment.
respectively, as shown in Figure 1. The results suggest that EAMP had
Acute Oral Toxicity Study a higher level of phenolic and flavonoidal contents as compared to
other extracts of B. vulgaris extracts root.
Acute toxicity study of B. vulgaris root extract was performed
on the albino rats according Lorke’s method [15].
Preliminary Phytochemical Screening
The preliminary phytochemical screening were carried out on
of B. vulgaris root as described by [18].
Estimation of total phenolic content [19] and total flavonoid
content [20]
The Total Phenolic Content (TPC) of extracts was measured
using the Folin-Ciocalteu method [20].
In vitro antioxidant by 2, 2-diphenyl-1-picrylhydrazyl scavenging
assay [21]
In vitro antioxidant by 2, 2-diphenyl-1-picrylhydrazyl
scavenging activity assay was monitored using [21] method.
In vivo Antioxidant
Figure 1: Comparative study of Total Phenolic content and Total
In vivo antioxidant activity was determined by [38].
Flavonoid content of various extracts of Beta vulgaris root
Lipid Peroxidation Assay (Thiobarbituric Acid Reactive
The values were expressed as mean ± standard error of mean; *P <
Substances) [22]
0.05, **P < 0.01 versus control. ns: Not significant
Lipid Peroxidation Assay thiobarbituric acid reactive
substances (TBARS) [22]. In Vitro Antioxidant Activity against 2, 2-Diphenyl-1-
Picrylhydrazyl
Superoxide Dismutase Assay [23]
The percentage of scavenging effect of DPPH on different
This was estimated by the reaction mixture which contained
extracts of Beta vulgaris root with a concentration of 1, 5, 10, 25,
0.1 mL of phenazine methosulfate (186 μL), 1.2 mL of sodium
50, and 100 μg/mL was compared with ascorbic acid as shown in
pyrophosphate buffer (0.052 mL; pH 7.0), 0.3 mL of the supernatant
Figure 2 and found dose-dependent inhibitory antioxidant potential
after centrifugation (1,500 × g for 10 min followed by 10,000 × g for
against DPPH. Positive DPPH test suggests that the samples were
15 min) of homogenate was added to the reaction mixture. Enzyme
free radical scavengers. The IC50 values of CEBVR, MEBVR, EABVR,
reaction was initiated by adding 0.2 mL of NADH (780 μM) and
and ascorbic acid in DPPH assay were 85.97, 67.85, 46.06, and 23.74
stopped after 1 min by adding 1 mL of glacial acetic acid. The amount
μg/mL, respectively, as shown in Figure 3. Among all these extracts,
of chromogen formed was measured by recording color intensity at
the ethyl acetate extract was found better DPPH scavenging activity
560 nm. Results were expressed in units/mg protein.
with a minimum IC50 value of 46.06 μg/mL as compared to other
Catalase Assay [24] extracts.
It was determined with reaction solution contained 2.5 mL of
0.05 M phosphate buffers (pH 8.3), 0.7 mL of 0.2 M H2O2 and 0.1 mL

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Table 2: Effect of Beta vulgaris Root on Oxidative Markers in Rat

Brain

Treatment Control 100 200 300 400

(mgml-1) (mgml-1) (mgml-1) (mgml-1)
TBARS 2.51 + 4.85 ± 0.76 4.81 ± 1.04 4.46 ± 1.11 4.16 ± 1.2
mol/min/ 0.45
mg protein
SOD U/mg 12.3 + 21 15.1 ± 4.5 23.7 ± 12.1 ± 2.9 12.3 ±
protein 9.6a, b 0.98
CAT (U/ 1.89 ± 2.54 ± 0.77 1.67 ± 0.87 1.2 ± 0.89 2.05 ±
mm) 0.55a 0.98
GSH 2.61 ± 15.4 ± 1.6 15.6 ± 2.5 14.3 ± 2.2 15.4 ± 1.6
Figure 2: Effect of Beta vulgaris root extracts on percentage of (Umol/g 0.92**
inhibition against 2, 2-diphenyl-1-picrylhydrazyl. tissue)

The values were expressed as mean ± standard error of mean (n =
6). CEBVR, MEBVR, and EABVR were the extracts of chloroform,
methanol, and ethyl acetate extract of Beta vulgaris extracts root
respectively.
The oxidative stress markers as lipid peroxidation (TBARS),
SOD, CAT, GSH in rat brain homogenate was evaluated. EABVR at 200
and 400 mg/kg showed significantly (P < 0.01) and dose-dependently
increased the level of antioxidant enzymes such as SOD, CAT, and GSH
in brain tissue as compared to control rats as shown in Figure 3.
However, EAMP (100 mg/kg) was produced no significant changes
in antioxidant enzymes in rat brain tissue. In addition, EABVR at 400
mg/kg dose was significantly (P < 0.01) reduced the lipid peroxidation
that is TBARS level to 4.85 ± 0.76 as compared to control rats of 2.51
+ 0.45 nM/min/mg protein. No significant change in TBBVR level was
observed for EABVR (100 and 200 mg/kg) treated rat.

Figure 3: Estimation of IC50 value of various extracts of B. vulgaris
root leaves against 2, 2-diphenyl-1-picrylhydrazyl.
The values were expressed as mean ± standard error of mean (n =
3). **P < 0.01 versus standard. CEBVR, MEBVR, and EABVR were
the extracts of chloroform, methanol, and ethyl acetate extract of B.
vulgaris root, respectively.
In Vivo Antioxidant Activity of Beta vulgaris Root Leaves on Rat
Effect of B. vulgaris root leaves on body weight and brain weight.
No significant changes were observed in body weight of treated and
control rats before, and after the administration of the ethyl acetate
extract of B. vulgaris root for 7 days. Similarly, no considerable
differences in the wet weight of the whole brain between control and
EAMP treated rats as shown in Table 1.
Table 1: Effect of EABVR on Body Weight and Brain Weight in Rats
DISCUSSION
Natural antioxidants that are ubiquitous in fruits, vegetables,
and medicinal plants have received great attention in recent times
since they are effective and lesser toxic than synthetic antioxidants
[26]. Polyphenols and flavonoids act as an antioxidant agent by the
property of hydrogen atom donators, singlet oxygen scavengers, and
free radical scavenger [27,28]. Again, flavonoids have been reported
as inhibitors of lipid peroxidation [29] and prevent oxidative damage
[30]. The present investigation observed that the TPCs of crude
extracts of B. vulgaris root varied from 8.43 ± 1.03 to 15.64 ± 1.31 mg
GAE/g, whereas the TFCs varied from 0.75 ± 0.10 to 1.97 ± 0.47 mg
QUE/g. All the extracts of B. vulgaris root have a significant amount
of phenolic and flavonoid components. However, the EABVR extract
possessed the higher amount of phenolic and flavonoid content as
compared to other extracts. They can be ranked as EABVR > MEBVR >
CEBVR. The presence of flavonoids and phenolic components in ethyl
acetate extract of B. vulgaris root substantiate the claim for its free
radical scavenging activity and further attenuate the progression of
oxidative stress induced diseases.

Group Treatment DOSE Before After Wet Antioxidants may offer resistance against the oxidative stress
mg/kg Treatment Treatment Brain by scavenging the free radicals, inhibiting the lipid peroxidation, and
Weight by many other mechanisms and thus prevent diseases. Antioxidant
(g) activity is expressed in terms of IC50, and a lower IC50 value
corresponds to a larger scavenging activity [31]. DPPH is a compound
I Control D.W 178.1 + 181.1 + 1.76 + consists of a nitrogen free radical, which is easily quenched by a free
3.1 1.0* 0.03 radical scavenger. DPPH radicals are reduced into a non-radical form
II EABVR 100 180.2 + 188.2 + 177 + (DPPH-H) in the presence of a proton radical scavenger or hydrogen
2.4* 1.0 * 0.02 donating antioxidants [32]. In fact, DPPH radical has an absorbance
at 517 nm, which disappears after acceptance of an electron or
III EABVR 200 181.1 + 186 + 1.8* 1.71 + hydrogen radical from an antioxidant compound to become a stable
3.6* 0.02* diamagnetic molecule [33]. The effect of free radical scavenging
IV EABVR 400 182.1 + 189.6 + 1.61 + activity of B. vulgaris root extracts on DPPH radicals is thought to be
3.1* 0.7 0.03 due to their hydrogen donation ability of polyphenols. In the present
study, the extracts showed a dose dependent elevation in DPPH
All value were expressed as mean + SEM (n = 6), where *P<0.05
scavenging activity of which EABVR showed lower IC50 than others.
versus control. EABVR : Ethyl acetate extract of B.V; SEM: Standard
A similar study revealed that the aqueous extract of B. vulgaris root
Error of Mean. DW: Distilled water
showed a significant IC50 value (136 ± 0.003) but lower than ascorbic
acid [34].
Lipid peroxidation is a complex process occurring in aerobic
cells which reflects the interaction between molecular oxygen and

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polyunsaturated fatty acids. The radicals which are responsible for
lipid peroxidation, that also causes food deterioration, ageing of
organisms, and cancer promotion. These free radicals associated
with other relative species cause the oxidation of biomolecules
(e.g., Protein, amino acids, lipid, and DNA), which leads to cell injury
and death [3]. Lipid peroxidation in biological systems has long
been thought to be a toxicological phenomenon that can lead to
various pathological consequences [35]. The end products of lipid
peroxidation are reactive aldehydes, such as 4-hydroxyl non-enal
and Malondialdehyde (MDA), many of which are highly toxic to cells
[36]. In addition, the MDA can react with biomolecules and exert
cytotoxic, genotoxic, and neurodegenerative disorders. However, the
endogenous antioxidants such as SOD and CAT are susceptible to
oxidative changes. The enzyme SOD removes the superoxide anion,
while CAT, [37] a heme protein catalyses the reduction of H2O2 [38] and
protects the tissues from highly reactive hydroxyl radicals that could
be generated from H2O2. Indeed, GSH provides the first line of defense
for the body by scavenging ROS. Oxidative stress readily oxidizes
GSH non-enzymatically to glutathione disulfide by electrophilic
substances such as free radicals and ROS [39] and causes depletion of
GSH level by inhibiting glutamate-cystine antiporter [40]. Our results
showed that inhibition of MDA formation increases with increase in
dose of EAMP that is a reduction in TBARS level. In addition, there is
a significant increase of antioxidant enzymes such as SOD and CAT
and non-enzymatic GSH level in rat brain homogenate, which is due
to the linkage between phenolics and antioxidant enzymatic and non-
enzymatic activity [40,41] Moreover, literature reveals that B. vulgaris
root showed the presence of mimosine, terpenoids, flavonoids,
glycosides, alkaloids, quinines, phenols, tannins, saponins, coumarins,
d-xylose and d-glucuronic acid 4-O-(3,5-dihydroxybenzoic acid)-β-D-
glucoronide, [11] might have contributed to the antioxidant activity.
Several studies were demonstrated by in vitro and in situ models
that, certain flavonoids e.g., quercetin and catechins are capable of
penetrate through the blood-brain barrier. Earlier reports found
the presence of phenolics, flavonoids, and tannins were found to
possess the antioxidant property and attenuate cell death induced by
oxidative stress, which supported our present findings [40-48].
In summary, the present study revealed that the EABVR showed
the highest level of total phenolic, as well as flavonoid compounds,
and were capable of initiating, quenching free radicals to terminate
the radical chain reaction, and acting as reducing agents. Moreover,
a significant and linear relationship was found between the
antioxidant activity and phenolic content, indicating that phenolic
compounds could be major contributors to antioxidant activity. In
addition, it has demonstrated that EABVR had a greater potential
of the inhibitory effect on cell lipid peroxidation and improved the
antioxidant enzymes in rat brain homogenate. The antioxidant
and biological activities might be due to the synergistic action of
bioactive compounds present in them. The observation of this current
investigation strongly suggests the potential antioxidant activity of
EABVR leaves potentiates the neuroprotective effect by stimulating
brain enzymes. Hence, it may be used as a weapon in various
neuroinflammatory and neurodegenerative diseases. Furthermore,
studies are warranted for the isolation and the identification of
individual phenolic compounds; and also in vivo studies are needed
for understanding their mechanism of action as an antioxidant better.
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